ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We Pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context, Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data Suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1. (C) 2009 Elsevier Inc. All rights reserved.